The SH3 domain-containing protein Nbp2 targets the Ptc1 type 2C
Ser/Thr phosphatase to the mitogen-activated protein kinase
module.
James Mapes (1), Irene Ota (2)
(1) Chemistry and Biochemistry, University of Colorado, UCB 215,
Boulder, CO 80309, US;
(2) Myriad Proteomics, Salt Lake City, UT
The type 2C Ser/Thr phosphatase, Ptc1, negatively regulates the yeast
high osmolarity glycerol (HOG) pathway by inactivating the Hog1 mitogen-activated protein kinase (MAPK). We provide the first evidence that this
class of phosphatase inactivates MAPK via a novel scaffold protein,
Nbp2. First, phenotypic data indicated that Nbp2 like Ptc1 was a
negative regulator. Previously, we showed that deletion of PTC1
together with PTP2, encoding a protein tyrosine phosphatase, was
nearly lethal due to Hog1 hyper-phosphorylation and activation. Similar
to ptc1∆ ptp2∆, the nbp2∆
ptp2∆ strain showed severe HOG1-dependent growth
defects. In addition, the hyperactive MEKK allele in this pathway was
lethal in ptc1∆ and nbp2∆, but not in
ptc1∆ hog1∆ and nbp2∆ hog1∆.
Second, biochemical data showed Nbp2 was a negative regulator. Similar
to ptc1∆, the nbp2∆ strain exhibited high
basal Hog1 kinase activity, and an inability to inactivate Hog1 during
adaptation. The molecular mechanism by which Nbp2 acts as a negative
regulator was also explored. We hypothesized that Nbp2 recruited Ptc1 to
the Pbs2 MEK-Hog1 complex. Consistent with Nbp2 mediating Ptc1
interaction with Pbs2, Ptc1 bound Pbs2 in an Nbp2-dependent manner. The
Nbp2 N-terminal domain bound Ptc1, while the Nbp2 SH3 domain bound Pbs2
via its Pro rich sequence. Thus, Nbp2 acts as a scaffold protein,
recruiting the Ptc1 phosphatase to the MEK-MAPK module.
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